Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
2001-05-01
2003-07-22
Nguyen, Nam (Department: 1743)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
C204S461000, C204S603000, C204S612000
Reexamination Certificate
active
06596140
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to electrophoretic separation systems for the analysis of bio-molecules, such as nucleic acids. More particularly, this invention relates to a multi-channel capillary electrophoresis device and method wherein the distortion of a sample zone exiting from the end of a channel is controlled thereby resulting in enhanced detectability of such sample zone.
REFERENCES
Dovichi et al., U.S. Pat. No. 5,439,578 (1995).
Grossman and Colburn, Capillary Electrophoresis Theory and Practice, Chapter 1, Academic Press (1992).
Grossman, U.S. Pat. No. 5,374,527 (1994).
Holman, Heat Transfer, Fourth Edition, McGraw-Hill (1976).
Kambara et al., U.S. Pat. No. 5,192,142 (1993).
Madabhushi et al., U.S. Pat. No. 5,552,028 (1996).
Sambrook et al., eds., Molecular Cloning: A Laboratory Manual, Second Edition, Chapter 5, Cold Spring Harbor Laboratory Press (1989).
Takahashi et al., U.S. Pat. No. 5,529,679 (1996).
Woolley, et al., Ultra-high-speed DNA fragment separations using microfabricated capillary array electrophoresis chips, Proc. Natl. Acad. Sci., vol. 91, pp. 11348-11352, Nov. 1994, Biophysics.
BACKGROUND OF THE INVENTION
Electrophoretic separations of bio-molecules are critically important in modern biology and biotechnology, comprising an important component of such techniques as DNA sequencing, protein molecular weight determination, genetic mapping, and the like. A particularly preferred electrophoresis format is capillary electrophoresis (CE), where the electrophoresis is performed in a channel, such as a capillary tube or a groove in a microfabricated chip, wafer or plate, having a small internal diameter. Capillary electrophoresis results in enhanced separation performance over traditional slab-based formats because the superior ability of the narrow-bore capillary to tolerate resistive heating allows for high electrical fields to be employed thereby resulting in fast separations in which sample diffusion is minimized.
In traditional CE systems, detection of a sample subsequent to separation is performed during electrophoresis while the sample is still located inside the channel (referred to as “on-channel” detection). Thus, in a common capillary tube arrangement, any excitation light required to excite the sample and any emission light coming from the sample must be transmitted through the wall of the capillary tube. A drawback of this approach is that the fused silica capillary tubes often used in CE have numerous surfaces to reflect or scatter light. Problems associated with light scattering are particularly problematic when it is desired to detect fluorescence from samples located in a plurality of closely-spaced capillary tubes by fluorescence because the scattered emmission light from one capillary tube can interfere with the detection of samples in neighboring capillary tubes.
One approach to solving the problem of on-channel detection has been to detect a sample after the sample emerges from the capillary (referred to as “off-channel” detection). In one type of arrangement, such off-channel detection takes place in a detection cell positioned downstream of the capillary tube outlets. Typically, the detection cell is configured to exhibit superior optical characteristics, e.g., a flat quartz chamber. In one class of these systems, a “sheath flow” of liquid is used to transport the sample from the outlet of the CE capillary tube to a detection zone at which detection of the sample takes place (Takahashi; Dovichi). A drawback of sheath flow systems is that, in order to avoid distortion of a sample zone in the detection cell, precise control of the flow rate of the sheath flow liquid is required. A second drawback of sheath flow systems is that the pressure used to drive the flow of the sheath flow liquid can cause back flow of the separation medium in the separation capillary tube, thereby negatively impacting resolution.
In another class of off-channel detection systems, a sample zone is transported from the outlet of a CE capillary tube to a detection zone located in a detection cell by electrophoresis under the influence of the same voltage difference used to conduct the electrophoretic separation (Takahashi). However, because of the larger cross-sectional area within the detection cell as compared to the lumen of the capillary tube, the electric field diverges at the capillary tube outlet causing a distortion of the sample zone as it enters and traverses the detection zone. Unchecked, such distortion can result in a severe loss of spatial resolution between adjacent sample zones exiting a single capillary tube and/or between sample zones exiting adjacent capillary tubes. This loss of spatial resolution tends to reduce the detectability of neighboring sample zones.
SUMMARY OF THE INVENTION
Generally, the present invention relates to a device and method for electrophoretically transporting a sample zone from an electrophoresis channel, via an outlet end thereof, into a detection zone or chamber downstream of the channel, where the distortion of the sample zone is controlled in a fashion permitting enhanced detectability.
The various embodiments of the device and method of the present invention find particular application in automated polynucleotide sequencing systems employing fluorescence detection and a plurality of separation channels (e.g., capillary electrophoresis tubes or microfabricated (e.g., etched) channels in a plate).
More particularly, in one of its aspects, the present invention relates to an analyte separation device, such as a CE tube or plate device, including (i) a plurality of separation channels, with each channel comprising an inlet end and an outlet end; (ii) a detection zone proximate the outlet ends; and (iii) at least one excitation-beam pathway extending through at least a portion of said detection zone. In an embodiment of the device, two or more of the channels have a variation region, in which the channel cross-sectional area varies (e.g., progressively increases), in the vicinity of (i.e., near and/or along) their outlet ends.
The device can further include an excitation-beam source for directing an excitation beam along said excitation-beam pathway(s). Any suitable beam source can be employed. In an embodiment of the invention, the beam source is a laser. The present invention contemplates for example (without limitation): a side-entry beam arrangement, a scanning or fanned beam (or other broad) illumination arrangement, and/or an up-channel (axial) illumination arrangement. In an embodiment of the latter, sample excitation takes place at, or not far beyond, the variation region of each channel.
In another of its aspects, the present invention relates to an analyte separation device, such as a CE tube or plate device, having an off-channel detection arrangement. In one embodiment, the device includes (i) a plurality of separation channels, each channel comprising an inlet end and an outlet end; (ii) a detection chamber, or zone, proximate the outlet ends; and (iii) an excitation-beam pathway extending through at least a portion of the detection chamber, with the pathway (a) being located on a side of the outlet ends opposite the inlet ends (i.e., downstream of the outlet ends) and (b) extending along a plane defined by the channels (e.g., a side-entry arrangement). Two or more of the channels are provided with a variation region, in which the channel cross-sectional area varies, along a region near respective outlet ends.
According to one embodiment, the cross-sectional area of the variation region increases along a direction extending from the inlet end to the outlet end. In another embodiment, the cross-sectional area of the variation region decreases along a direction extending from the inlet end to the outlet end. In a further embodiment, the variation region comprises a first portion in which the cross-sectional area decreases along a direction extending from the inlet end to the outlet end, and a second portion in which the cross-sectional area increases along said direction, with the second
Nordman Eric S.
Reel Richard T.
Applera Corporation
Harness & Dickey & Pierce P.L.C.
Nguyen Nam
Noguerola Alex
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